Topoisomerases are ubiquitous cellular enzymes required for controling DNA topology during DNA synthesis by removing free supercoiling. On the molecular level, topoisomerase II (Top2) cuts both strands of DNA duplex using its tyrosine residue to generate transient double-strand breaks (DSBs) and form the Top2 cleavage complex, in which Top2 is covalently linked to the 5′terminus of the DSB via a tyrosyl phosphodiester bond. Continuous DNA transcription and replication requires a re-ligation of the DSB at the end of each catalytic cycle such that a dynamic equilibrium is established between DNA and the Top2cc. However, when the transient Top2 cleavage occurs near a pre-existing DNA damage the re-ligation is stalled and the cleavage complex becomes abortive. Clinically important Top2 poisons work by this mechanism as they bind to and stabilize the Top2cc to prevent re-ligation, resulting in the accumulation of abortive Top2cc. Such DNA damages are often repaired by cellular DNA repair enzymes.
Tyrosyl-DNA phosphodiesterase II (TDP2), a DNA repair enzyme, specifically repairs Top2-mediated DNA damages, including the abortive Top2cc trapped by Top2 poisons (Ledesma, F. C., et al., Nature 2009, 461, 674-U125). As a result, the normal cellular function of TDP2 renders cancer cells resistant to Top2 poisons, a major class of drugs widely used to treat cancers such as testicular cancer, lung cancer, lymphoma, leukemia, neuroblastoma, and ovarian cancer. This is supported by observations both in cultured cells and animal models that the lack of TDP2 leads to enhanced cellular sensitivity to DNA breaks induced by Top2 poisons (Ledesma, F. C., et al., Nature 2009, 461, 674-U125; Zeng, Z. H., et al., J. Biol. Chem. 2011, 286, 403-409; Gomez-Herreros, F., et al., Nat. Genet. 2014, 46, 516-521; Gomez-Herreros, F., et al., PLoS Genet. 2013, 9, e1003226; and Maede, Y., et al., Mol. Cancer. Ther. 2014, 13, 214-20). Importantly, up-regulation of TDP2 transcription through a gain-of-function p53 mutation has indeed been linked to Top2 poison resistance in human lung cancer (Do, P. M., et al., Genes Dev. 2012, 26, 830-45). These observations strongly suggest that inhibiting TDP2 could sensitize cancer cells towards clinical Top2 poisons.
TDP2 inhibition, however, is underexplored and poorly understood. Since its discovery in 2009 (Ledesma, F. C., et al., Nature 2009, 461, 674-U125), efforts in biochemistry and crystallography have generated critical knowledge to allow basic understanding on TDP2 active site, mode of substrate recognition, enzyme kinetics, and mechanism of catalysis (Gao, R., et al., J. Biol. Chem. 2012, 287, 30842-30852; Schellenberg, M. J., et al., Nat. Struct. Mol. Biol. 2012, 19, 1363-1371; and Shi, K., et al., Nat. Struct. Mol. Biol. 2012, 19, 1372-1377). These studies support a single-metal catalytic mechanism characteristic of the exonuclease-endonuclease-phosphatase (EEP) nuclease superfamily. According to this mechanism, TDP2 cleaves the 5′ phosphotyrosine adduct through a hydrolytic reaction promoted by one Mg2+ ion and a few key residues at active site. Particularly important to catalysis are residues D262, E152, H351 and S229, as single mutation of any of them was shown to abrogate the catalytic activity of hTDP2 (Schellenberg, M. J., et al., Nat. Struct. Mol. Biol. 2012, 19, 1363-1371). Importantly, crystal structures of TDP2 bound to DNA substrates revealed a deep, narrow groove that selectively accommodates the 5′ end of single-stranded DNA (Withoff, S., et al., Anticancer Res. 1996, 16, 1867-1880 and Shi, K., et al., Nat. Struct. Mol. Biol. 2012, 19, 1372-1377). Nevertheless, efforts in TDP2 inhibition to date have been largely limited to random screening of compound libraries using biochemical assays (Laev, S., et al., Bioorg. Med. Chem. 2016, 24, 5017-5027). Hits generated typically lack desired drug like properties and fit the profiles of pan-assay interference structure (PAINS) (Baell, J. B., et al. J. Med. Chem. 2010, 53, 2719-2740).
Certain specific compounds that were identified through a high-throughput-screening (HTS), selectively inhibited hTDP2 in nanomolar range, presumably by occupying the DNA binding groove of TDP2 and blocking DNA substrates from coming into TDP2 active site (Raoof, A., et al., J. Med. Chem. 2013, 56, 6352-6370; and Hornyak, P., et al., Biochem. J. 2016, 473, 1869-1879). However, these biochemical inhibitors show only weak efficacy in cancer cells (Marchand, C., et al., ACS Chem. Biol. 2016, 11, 1925-1933).
Currently, there is a need for TDP2 inhibitors (e.g. improved TDP2 inhibitors) that are effective in sensitizing cancer cells to allow Top2 poisons to be used at lower and safer doses.